Functional cloning of SPIN-2, a nuclear anti-apoptotic protein with roles in cell cycle progression

Abstract

The balance between hematopoietic cell viability and apoptosis is regulated by exogenous growth factors, however, the molecular mechanisms by which these trophic factors exert their effects remain obscure. A functional retroviral cDNA librarybased screen was employed to identify genes that prevent growth factor withdrawal-mediated apoptosis in the myeloid progenitor cell 32Dcl3. This approach identified three classes of genes: those with known roles in apoptosis (bcl-X L and ornithine decarboxylase); genes previously identified but not linked directly to apoptotic signaling (O-linked N-acetylglucosamine transferase); and a previously uncharacterized gene we termed SPIN-2. In 32Dcl3 cells, expression of exogenous SPIN-2 provides 25% protection from apoptosis following growth factor withdrawal compared to controls which show ෂ1-2% survival. SPIN-2 overexpression slows cell growth rates and increases the percentage of cells in G 2 /M (32% vs control cells at 12%).Immunolocalization studies indicate that myc-epitope tagged SPIN-2 proteins, which retain their anti-apoptotic function, reside in the nucleus, whereas a C-terminal deletion mutant that loses its anti-apoptotic activity is located in the cytoplasm. These studies suggest that SPIN-2 is a novel nuclear protein that functions to regulate cell cycle progression and this activity is related to the inhibition of apoptosis following the removal of essential growth factors.

Potent Anti-tumor Effects of an Active Site Mutant of Human Manganese-Superoxide Dismutase

“…For the generation of retrovirally transduced A549 cells, Phoenix cells at ϳ40% confluency were transfected using calcium phosphate with 8 g of empty vector, wild-type Mn-SOD, H30N Mn-SOD, or GFP (pLPCX only) and incubated at 37°C (27). The same protocol was used for both the pBMN-IRES-GFP and pLPCX constructs.…”

“…The Mn-SOD cDNAs were excised from the pcDNA 3.1 plasmid using EcoRI and NotI and subcloned into both of these retroviral vectors. Phoenix-packaging cells were used for the production of infectious retroviruses (27). A549 cells were utilized for the retroviral transduction experiments and were cultured in Ham's modified F12K media containing 10% fetal bovine serum.…”

“…Retroviral Constructs, Transductions, and Cell Growth-Wild-type (WT) Mn-SOD and H30N Mn-SOD mutant cDNAs were subcloned from the pcDNA 3.1 plasmid into a pBMN-IRES-eGFP retroviral expression vector (27) and into the puromycin selectable retroviral expression vector pLPCX (Clontech Corp.). The Mn-SOD cDNAs were excised from the pcDNA 3.1 plasmid using EcoRI and NotI and subcloned into both of these retroviral vectors.…”

“…Animal Model of Tumorigenesis-NOD/SCID mice (28) were injected subcutaneously with 2 ϫ 10 6 retrovirally transduced A549 cells overexpressing empty pBMN-IRES-GFP vector (27), wild-type Mn-SOD, or the H30N mutant in pBMN-IRES-GFP (n ϭ 7 per group). Flow cytometry of GFP fluorescence was performed prior to injection to determine the transduction efficiency, which was between 88 and 92%.…”

Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis

“…Nucleic acid libraries provide some of the most versatile tools for functional analysis of genomes, individual proteins or complexes [1][2][3][4][5][6][7][8][9][10][11][12][13][14] . These libraries can be constructed using either biologically derived or chemically synthesized nucleic acids as substrates.…”